The phloem-feeding whitefly Bemisia tabaci is a serious pest to a broad range of host plants, including many economically important crops such as tomato. These insects serve as a vector for various devastating plant viruses. It is known that whiteflies are capable of manipulating host-defense responses, potentially mediated by effector molecules in the whitefly saliva. We hypothesized that, beside putative effector proteins, small RNAs (sRNA) are delivered by B. tabaci into the phloem, where they may play a role in manipulating host plant defenses. There is already evidence to suggest that sRNAs can mediate the host-pathogen dialogue. It has been shown that Botrytis cinerea, the causal agent of gray mold disease, takes advantage of the plant sRNA machinery to selectively silence host genes involved in defense signaling. Here we identified sRNAs originating from B. tabaci in the phloem of tomato plants on which they are feeding. sRNAs were isolated and sequenced from tomato phloem of whitefly-infested and control plants as well as from the nymphs themselves, control leaflets, and from the infested leaflets. Using stem-loop RT-PCR, three whitefly sRNAs have been verified to be present in whitefly-infested leaflets that were also present in the whitefly-infested phloem sample. Our results show that whitefly sRNAs are indeed present in tomato tissues upon feeding, and they appear to be mobile in the phloem. Their role in the host-insect interaction can now be investigated.

Figure 4: Detection of candidate transferred whitefly sRNAs in the nymph and leaflet samples by stem-loop RT-PCRs. Expression of sRNAs in: leaflet first infested with whiteflies but with adults and nymphs subsequently removed (LW), whitefly nymphs (WN), control leaflet (LC) or leaflet with eggs (LE). (A) sRNA #13120, a specific band is detected in LW and WN but not in LC and LE. (B) #18833, a specific band is detected in LW and WN but not in LC and LE. Similar results for #13120 and #18833 were obtained in four biological replicates. (C) #3182, a specific band is detected in LW and WN but not in LC and LE in three out of four replicates. One biological replicate showed also a band in LC and LE. Molecular Mass (MM), GeneRuler Ultra Low Range DNA ladder (Thermo Scientific).

Mentions:
sRNA sequencing of the phloem samples identified the presence of mobile whitefly-originating sRNAs. We selected three whitefly candidate sRNAs from the final list (Table 1) for validation using stem-loop PCR on leaflets. The criteria for selecting these specific candidates from the sRNAseq data were (1) a length between 23 and 24 nt (Figure 2), (2) present among highest counts in nymphs, (3) presence in whitefly-infested leaflets (LW), in phloem from leaflets infested with whiteflies (PW) and in the B. tabaci nymph (WN) sample, while absent in the control leaflet (LC), absent in leaflets with only eggs (LE), and absent in the phloem control sample, and finally (4) preferably matching an insect-like or an unknown small RNA in the miRBase. From the three selected sRNAs, sRNA #13120, and #18833 were annotated as insect miR305 and miR1175-3p, respectively, using the miRBase (Kozomara and Griffiths-Jones, 2014). sRNAs #13120 (Figure 4A) and #18833 (Figure 4B) were present in nymph and were found back in three out of four LW samples while being absent in all LC and LE samples. sRNA #3182 did not provide a match in the miRBase but could be amplified in nymph and all infested leaf samples, however it was found in one out of four control samples. Overall, whitefly sRNAs could be detected within the leaflet samples (Figure 4C) on which nymph feeding took place. Since two out of three candidate small RNAs were found exclusively in the infested samples PW and LW, we conclude that whiteflies transfer small RNAs to the phloem, which then have the potential to move.

Figure 4: Detection of candidate transferred whitefly sRNAs in the nymph and leaflet samples by stem-loop RT-PCRs. Expression of sRNAs in: leaflet first infested with whiteflies but with adults and nymphs subsequently removed (LW), whitefly nymphs (WN), control leaflet (LC) or leaflet with eggs (LE). (A) sRNA #13120, a specific band is detected in LW and WN but not in LC and LE. (B) #18833, a specific band is detected in LW and WN but not in LC and LE. Similar results for #13120 and #18833 were obtained in four biological replicates. (C) #3182, a specific band is detected in LW and WN but not in LC and LE in three out of four replicates. One biological replicate showed also a band in LC and LE. Molecular Mass (MM), GeneRuler Ultra Low Range DNA ladder (Thermo Scientific).

Mentions:
sRNA sequencing of the phloem samples identified the presence of mobile whitefly-originating sRNAs. We selected three whitefly candidate sRNAs from the final list (Table 1) for validation using stem-loop PCR on leaflets. The criteria for selecting these specific candidates from the sRNAseq data were (1) a length between 23 and 24 nt (Figure 2), (2) present among highest counts in nymphs, (3) presence in whitefly-infested leaflets (LW), in phloem from leaflets infested with whiteflies (PW) and in the B. tabaci nymph (WN) sample, while absent in the control leaflet (LC), absent in leaflets with only eggs (LE), and absent in the phloem control sample, and finally (4) preferably matching an insect-like or an unknown small RNA in the miRBase. From the three selected sRNAs, sRNA #13120, and #18833 were annotated as insect miR305 and miR1175-3p, respectively, using the miRBase (Kozomara and Griffiths-Jones, 2014). sRNAs #13120 (Figure 4A) and #18833 (Figure 4B) were present in nymph and were found back in three out of four LW samples while being absent in all LC and LE samples. sRNA #3182 did not provide a match in the miRBase but could be amplified in nymph and all infested leaf samples, however it was found in one out of four control samples. Overall, whitefly sRNAs could be detected within the leaflet samples (Figure 4C) on which nymph feeding took place. Since two out of three candidate small RNAs were found exclusively in the infested samples PW and LW, we conclude that whiteflies transfer small RNAs to the phloem, which then have the potential to move.

The phloem-feeding whitefly Bemisia tabaci is a serious pest to a broad range of host plants, including many economically important crops such as tomato. These insects serve as a vector for various devastating plant viruses. It is known that whiteflies are capable of manipulating host-defense responses, potentially mediated by effector molecules in the whitefly saliva. We hypothesized that, beside putative effector proteins, small RNAs (sRNA) are delivered by B. tabaci into the phloem, where they may play a role in manipulating host plant defenses. There is already evidence to suggest that sRNAs can mediate the host-pathogen dialogue. It has been shown that Botrytis cinerea, the causal agent of gray mold disease, takes advantage of the plant sRNA machinery to selectively silence host genes involved in defense signaling. Here we identified sRNAs originating from B. tabaci in the phloem of tomato plants on which they are feeding. sRNAs were isolated and sequenced from tomato phloem of whitefly-infested and control plants as well as from the nymphs themselves, control leaflets, and from the infested leaflets. Using stem-loop RT-PCR, three whitefly sRNAs have been verified to be present in whitefly-infested leaflets that were also present in the whitefly-infested phloem sample. Our results show that whitefly sRNAs are indeed present in tomato tissues upon feeding, and they appear to be mobile in the phloem. Their role in the host-insect interaction can now be investigated.